Bacterial Protein E.P.C (Expression, Purification and Characterization) Title: Expression, Purification, and Characterization of pGEM-gbr22 protein Introduction: In research these days, there are now a bountiful amount of methods to produce recombinant proteins by expressing, purifying, and characterizing a specific protein. E. coli has been proven to be an great host for production of a variety of proteins because of its relative abundance and low cost [1]. E. coli BL21 (DE3) has shown that it is also a good host for strains with a lack of both lon and ompT proteases [1]. Recombinant protein plasmids have often been tagged with a hexa-histidine affinity tag for purification of the protein, and a AMP resistant gene has often been inserted into the plasmid as well. The recombinant plasmid can then be placed into the BL21 (DE3) bacterial genome, and the bacteria can be manipulated to express the protein. Then, after protein expression, the bacterial cells could then be lysed (split), and the target protein could run through a column containing Ni-NTA beads/resin so that the hexa-histidine affinity tag would bind to the resin and the target protein would then be successfully isolated and purified [2]. And the UV-Vis spectroscopy can be used to estimate the concentration of the purified protein in mg/mL.
Then, to characterize the purified protein, both SDS- PAGE analysis and UV absorption spectrophotometry can be done. After staining the gel with a dye, the intensity of the bands relatively represents the amount of the protein present in each sample. Lastly, the purity of the sample can be estimated using SDS-PAGE [1].
Materials & Methods:
In the first part of the lab, bacteria expression, 25 ul of E. Coli [BL21 (DE3)] was added to two transformation tubes. Then 2 ul of pGEM-gbr22 protein, the plasmid that has antibiotic resistance, was mixed with a culture of bacteria and heat shocked at 45 degrees Celsius and then placed in ice for 2 minutes. SOC was added for growth and the culture was then spread onto Amp agar plates incubated overnight at 37 degrees Celsius that produced colonies of bacteria. A starter culture was grown with a single bacteria colony called the LB, and then 100 mg/ml of Ampicillin was added and placed into a shake incubator at 37 degrees Celsius for about 8 hours. The starter culture was then transferred into a larger culture with 100 mg/ml of additional ampicillin and LB and shake incubated for 24 hours. The culture was then centrifuged at 5000 rpm at 4 degrees Celsius for 10 minutes on the Allegra X-15 benchtop centrifuge in which a purple pellet of transformed cells was produced. 1X PBS and lysozyme were added to the pellet and vortexed to obtain an even suspension of cells and the tube was then frozen at -20 degrees Celsius.
In the purification part of the lab, the lysate was made and purified by Benzonase, centrifuged at 14,000 rpm for 20 minutes (supernatant - sample 2), and syringe filtered through a PES syringe filter. The Wash and Elution buffers were prepared. The mixture was further purified with 0.5 mL of Ni-NTA affinity through a Bio-rad Econo column and added to the supernatant. 1X PBS, 20 mM imidazole, and 250 mM imidazole were run through the column to produce waste, wash, and Elutions 1 and 2. Samples 1,2, 3, 4, 5, and 6 were collected this way. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of Elution 1 at 280 nm and 574 nm and then converted to mg/ml.
In the last part of the lab, Protein Charcterization, gel electrophoresis was conducted with 6X SDS-PAGE gel for the six samples of one of the lab partner's and samples 4, 5, and 6 of the other partner's. The tubes were placed on a heat block for 5 minutes at 5000 rpm and then electrophoresis was run for 25 minutes at 200 V. The first well of the gel contained the protein ladder (Thermo Fisher Scientific, Waltham, MA) and then followed by samples 1-6 and 4-6. Afterwards the gel was stained, wrapped in cellophane, and then dried. The gel results were then compared to the Fermentas molecular weight standard.
Results:
Figure 1: E. Coli BL21 (DE3) bacterial colonies after being incubated for approximately 24 hours in 37 degrees Celsius. The bacteria were manipulated to express an Ampicillin (Amp) resistant, purple, Great Barrier Reef coral gene (pGEM-gbr22) in the form of a protein.
Figure 2: E. Coli BL21 (DE3) bacterial colonies after being incubated for approximately 24 hours in 37 degrees Celsius with the antibiotic Ampicillin added to the agar. Hardly any colonies showed growth due to the antibiotic Amp that killed mostly all the bacteria.
Figure 3. "Fun" plate of bacteria. Sample was collected from a corner of the laboratory floor and incubated at 37 degrees Celsius for about 24 hours.
Figure 4: E. Coli BL21 (DE3) bacteria with expressed pGEM-gbr22 protein in a flask mixed with 25 ml LB broth and ampicillin media after incubation in a shaking water incubator at 37 degree Celsius for 24 hours.
Figure 5: Vortexed or spun down E. Coli BL21 (DE3) bacteria, from Allegra X-15 benchtop centrifuge, and with expressed pGEM-gbr22, protein pellet. The wet cell pellet weighed about 0.22 g.
Figure 6: Elution 1 (on the right) and Elution 2 (on the left) containing purified pGEM-gbr22 protein washed by Elution buffer solution from a Ni-NTA column using 250 mM imidazole and 1 M PBS solution.
Figure 7: Nanodrop spectrophotometer absorbance spectra of the first of two Elution 1 samples at 280 nm wavelength. Absorbance taken on 10 mm (1 cm) pathlength. Using Beer's Law (A=Ebc), an extinction coefficient of 39,100, a pathlength of 1 cm, and an absorbance of 0.727, the concentration (c) was calculated to be 1.86x10^-4 mg/ml.
Figure 8: Nanodrop spectrophotometer absorbance spectra of the second of two Elution 1 samples at 574 nm wavelength. Absorbance taken on 10 mm (1 cm) pathlength. Using Beer's Law (A=Ebc), an extinction coefficient of 39,100, a pathlength of 1 cm, and an absorbance of 1.400, the concentration (c) was calculated to be 3.58x10^-4 mg/ml.Discussion:
SAM_0866.JPG
Figure 9: Failed gel from SDS-PAGE of Fermentas PageRuler molecular weight standard. Samples 1-6, and a partner's Samples 4-6, are shown after running electrophoresis at 200 Volts for 25 minutes with gel cassette in position, which conducted a small amount of current without another cassette in the other spot.
proteinladder.PNG
Figure 10: Molecular weight standard for 4-20% TGS buffer when performing SDS-PAGE electrophoresis. Colored bands represent an estimate of the molecular weight (in kDa) of the proteins in a solution after SDS gel electrophoresis is done.
SAM_0880.JPG
Figure 11: Results of SDS gel electrophoresis run after 25 minutes at 200 Volts after destaining and before drying the gel. From left to right, the wells are represented by the Fermentas PageRuler molecular weights standard, Samples 1-6, and a partner's samples 4-6. Visible purple bands in the wells represent a distinguishable protein in the solution from which each of the samples came from. The darkness or the intensity of the bands represent the prevalence of protein in solution, and their position in relation to the molecular weights standards can determine the molecular weight of the protein.
SAM_0881.JPG
Figure 12: Same as Figure 12, but this is the image of gel after drying. The most distinguishable or prevalent bands are the S5 and S6 sample solutions, whcih represent the pGEM-gbr22 protein that we characterized.
Discussion:
The purification of the protein separated the protein from the overexpressed bacteria. Adding Lysozyme and Benzonase released the soluble materials from the cell. Then, centrifugation removed the large and insoluble cell materials. Using the affinity tag and Ni-NTA resin, all proteins except pGEM-gbr22 were filtered out.
Samples 1-6, which were used for gel electrophoresis, represented various samples taken during the protein expression and purification steps. Sample 1 contained the pGEM-gbr22 protein, which included both soluble and insoluble cell materials incubated and grown overnight before any filtration. Before sample 2 was taken, lysozyme was used to break down the cell wall and lyse the cell. Benzonase digested the DNA/RNA and reduced the viscosity of the mixture. Sample 2 contained everything except the large, insoluble protein particles filtered out by centrifugation. Sample 3 had all the proteins. A small amount of Imidazole was added to remove the proteins that were loosely bound to the resin, and then Sample 4 was collected. Sample 5 contained the purified pGEM-gbr22 proteinsdisbanded from the Ni-NTA due to the large amount of Imidazole added. And lastly, sample 6 contained the remant purified proteins that were not released in Sample 5. The wash buffer contained less concentrated Imidazole that washed away other proteins with two or three histidines while keeping the gbr22 protein with six histidines. The elution buffer contained more concentrated Imidazole. These two buffers were used in the experiment since the gbr22 protein has been modified to have six histidine residues attached to the C-terminus. This resulted in the histidine residues attaching to the nickel beads in the column. Adding a large amount of Imidazole made the histidine bind to the metal. The pGEM-gbr22 was released. Elution #1 was used for the Nanodrop spectrophotometer to find the absorbance at 280 nm and maximal wavelength (574 nm). After obtaining the extinction coefficient a journal article, the concentrations of the protein were calculated using Beer’s Law. The amount of purified protein in mg/mL was calculated at 280 nm and 574 nm with the voume and concentration of the protein. Using the dried gel and the Fermentas PageRuler protein ladder of Molecular Weight standards, the purified protein weighed approximately 29 kDa. In the end, Sample 5 contained the purified pGEM-gbr22 with about 60-65% purity.
Conclusions: By overexpressing pGEM-gbr22 protein in E. Coli BL21 (DE3) bacteria, the protein was then isolated and purified in a solution. Beer's Law was used to determine the concentration of the purified protein in solution, and by using SDS gel electrohoresis the molecular weight and purity was also determined in relation to the other proteins using a molecular weight standard. Through this experiment a protein was closely purified, but there will always be a chance that a minimal amount of a few other proteins in solution could remain. In the future, we could purify other specific proteins from a bacteria or virus and develop a drug and determine the target.
References: [1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M. Nat Methods. Protein production and purification. 2008, 5(2):135-46.
Title:
Expression, Purification, and Characterization of pGEM-gbr22 protein
Introduction:
In research these days, there are now a bountiful amount of methods to produce recombinant proteins by expressing, purifying, and characterizing a specific protein. E. coli has been proven to be an great host for production of a variety of proteins because of its relative abundance and low cost [1]. E. coli BL21 (DE3) has shown that it is also a good host for strains with a lack of both lon and ompT proteases [1]. Recombinant protein plasmids have often been tagged with a hexa-histidine affinity tag for purification of the protein, and a AMP resistant gene has often been inserted into the plasmid as well. The recombinant plasmid can then be placed into the BL21 (DE3) bacterial genome, and the bacteria can be manipulated to express the protein. Then, after protein expression, the bacterial cells could then be lysed (split), and the target protein could run through a column containing Ni-NTA beads/resin so that the hexa-histidine affinity tag would bind to the resin and the target protein would then be successfully isolated and purified [2]. And the UV-Vis spectroscopy can be used to estimate the concentration of the purified protein in mg/mL.
Then, to characterize the purified protein, both SDS- PAGE analysis and UV absorption spectrophotometry can be done. After staining the gel with a dye, the intensity of the bands relatively represents the amount of the protein present in each sample. Lastly, the purity of the sample can be estimated using SDS-PAGE [1].
Materials & Methods:
In the first part of the lab, bacteria expression, 25 ul of E. Coli [BL21 (DE3)] was added to two transformation tubes. Then 2 ul of pGEM-gbr22 protein, the plasmid that has antibiotic resistance, was mixed with a culture of bacteria and heat shocked at 45 degrees Celsius and then placed in ice for 2 minutes. SOC was added for growth and the culture was then spread onto Amp agar plates incubated overnight at 37 degrees Celsius that produced colonies of bacteria. A starter culture was grown with a single bacteria colony called the LB, and then 100 mg/ml of Ampicillin was added and placed into a shake incubator at 37 degrees Celsius for about 8 hours. The starter culture was then transferred into a larger culture with 100 mg/ml of additional ampicillin and LB and shake incubated for 24 hours. The culture was then centrifuged at 5000 rpm at 4 degrees Celsius for 10 minutes on the Allegra X-15 benchtop centrifuge in which a purple pellet of transformed cells was produced. 1X PBS and lysozyme were added to the pellet and vortexed to obtain an even suspension of cells and the tube was then frozen at -20 degrees Celsius.
In the purification part of the lab, the lysate was made and purified by Benzonase, centrifuged at 14,000 rpm for 20 minutes (supernatant - sample 2), and syringe filtered through a PES syringe filter. The Wash and Elution buffers were prepared. The mixture was further purified with 0.5 mL of Ni-NTA affinity through a Bio-rad Econo column and added to the supernatant. 1X PBS, 20 mM imidazole, and 250 mM imidazole were run through the column to produce waste, wash, and Elutions 1 and 2. Samples 1,2, 3, 4, 5, and 6 were collected this way. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of Elution 1 at 280 nm and 574 nm and then converted to mg/ml.
In the last part of the lab, Protein Charcterization, gel electrophoresis was conducted with 6X SDS-PAGE gel for the six samples of one of the lab partner's and samples 4, 5, and 6 of the other partner's. The tubes were placed on a heat block for 5 minutes at 5000 rpm and then electrophoresis was run for 25 minutes at 200 V. The first well of the gel contained the protein ladder (Thermo Fisher Scientific, Waltham, MA) and then followed by samples 1-6 and 4-6. Afterwards the gel was stained, wrapped in cellophane, and then dried. The gel results were then compared to the Fermentas molecular weight standard.
Results:
Figure 1: E. Coli BL21 (DE3) bacterial colonies after being incubated for approximately 24 hours in 37 degrees Celsius. The bacteria were manipulated to express an Ampicillin (Amp) resistant, purple, Great Barrier Reef coral gene (pGEM-gbr22) in the form of a protein.
Figure 2: E. Coli BL21 (DE3) bacterial colonies after being incubated for approximately 24 hours in 37 degrees Celsius with the antibiotic Ampicillin added to the agar. Hardly any colonies showed growth due to the antibiotic Amp that killed mostly all the bacteria.
Figure 3. "Fun" plate of bacteria. Sample was collected from a corner of the laboratory floor and incubated at 37 degrees Celsius for about 24 hours.
Figure 4: E. Coli BL21 (DE3) bacteria with expressed pGEM-gbr22 protein in a flask mixed with 25 ml LB broth and ampicillin media after incubation in a shaking water incubator at 37 degree Celsius for 24 hours.
Figure 5: Vortexed or spun down E. Coli BL21 (DE3) bacteria, from Allegra X-15 benchtop centrifuge, and with expressed pGEM-gbr22, protein pellet. The wet cell pellet weighed about 0.22 g.
Figure 6:
Elution 1 (on the right) and Elution 2 (on the left) containing purified pGEM-gbr22 protein washed by Elution buffer solution from a Ni-NTA column using 250 mM imidazole and 1 M PBS solution.
Figure 7: Nanodrop spectrophotometer absorbance spectra of the first of two Elution 1 samples at 280 nm wavelength. Absorbance taken on 10 mm (1 cm) pathlength. Using Beer's Law (A=Ebc), an extinction coefficient of 39,100, a pathlength of 1 cm, and an absorbance of 0.727, the concentration (c) was calculated to be 1.86x10^-4 mg/ml.
Figure 8: Nanodrop spectrophotometer absorbance spectra of the second of two Elution 1 samples at 574 nm wavelength. Absorbance taken on 10 mm (1 cm) pathlength. Using Beer's Law (A=Ebc), an extinction coefficient of 39,100, a pathlength of 1 cm, and an absorbance of 1.400, the concentration (c) was calculated to be 3.58x10^-4 mg/ml.Discussion:
Figure 9: Failed gel from SDS-PAGE of Fermentas PageRuler molecular weight standard. Samples 1-6, and a partner's Samples 4-6, are shown after running electrophoresis at 200 Volts for 25 minutes with gel cassette in position, which conducted a small amount of current without another cassette in the other spot.
Figure 10: Molecular weight standard for 4-20% TGS buffer when performing SDS-PAGE electrophoresis. Colored bands represent an estimate of the molecular weight (in kDa) of the proteins in a solution after SDS gel electrophoresis is done.
Figure 11: Results of SDS gel electrophoresis run after 25 minutes at 200 Volts after destaining and before drying the gel. From left to right, the wells are represented by the Fermentas PageRuler molecular weights standard, Samples 1-6, and a partner's samples 4-6. Visible purple bands in the wells represent a distinguishable protein in the solution from which each of the samples came from. The darkness or the intensity of the bands represent the prevalence of protein in solution, and their position in relation to the molecular weights standards can determine the molecular weight of the protein.
Figure 12: Same as Figure 12, but this is the image of gel after drying. The most distinguishable or prevalent bands are the S5 and S6 sample solutions, whcih represent the pGEM-gbr22 protein that we characterized.
Discussion:
The purification of the protein separated the protein from the overexpressed bacteria. Adding Lysozyme and Benzonase released the soluble materials from the cell. Then, centrifugation removed the large and insoluble cell materials. Using the affinity tag and Ni-NTA resin, all proteins except pGEM-gbr22 were filtered out.
Samples 1-6, which were used for gel electrophoresis, represented various samples taken during the protein expression and purification steps. Sample 1 contained the pGEM-gbr22 protein, which included both soluble and insoluble cell materials incubated and grown overnight before any filtration. Before sample 2 was taken, lysozyme was used to break down the cell wall and lyse the cell. Benzonase digested the DNA/RNA and reduced the viscosity of the mixture. Sample 2 contained everything except the large, insoluble protein particles filtered out by centrifugation. Sample 3 had all the proteins. A small amount of Imidazole was added to remove the proteins that were loosely bound to the resin, and then Sample 4 was collected. Sample 5 contained the purified pGEM-gbr22 proteinsdisbanded from the Ni-NTA due to the large amount of Imidazole added. And lastly, sample 6 contained the remant purified proteins that were not released in Sample 5.The wash buffer contained less concentrated Imidazole that washed away other proteins with two or three histidines while keeping the gbr22 protein with six histidines. The elution buffer contained more concentrated Imidazole. These two buffers were used in the experiment since the gbr22 protein has been modified to have six histidine residues attached to the C-terminus. This resulted in the histidine residues attaching to the nickel beads in the column. Adding a large amount of Imidazole made the histidine bind to the metal. The pGEM-gbr22 was released.
Elution #1 was used for the Nanodrop spectrophotometer to find the absorbance at 280 nm and maximal wavelength (574 nm). After obtaining the extinction coefficient a journal article, the concentrations of the protein were calculated using Beer’s Law. The amount of purified protein in mg/mL was calculated at 280 nm and 574 nm with the voume and concentration of the protein.
Using the dried gel and the Fermentas PageRuler protein ladder of Molecular Weight standards, the purified protein weighed approximately 29 kDa. In the end, Sample 5 contained the purified pGEM-gbr22 with about 60-65% purity.
Conclusions:
By overexpressing pGEM-gbr22 protein in E. Coli BL21 (DE3) bacteria, the protein was then isolated and purified in a solution. Beer's Law was used to determine the concentration of the purified protein in solution, and by using SDS gel electrohoresis the molecular weight and purity was also determined in relation to the other proteins using a molecular weight standard. Through this experiment a protein was closely purified, but there will always be a chance that a minimal amount of a few other proteins in solution could remain. In the future, we could purify other specific proteins from a bacteria or virus and develop a drug and determine the target.
References:
[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M. Nat Methods. Protein production and purification. 2008, 5(2):135-46.
[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: ProteinExpression .http://www.embl.de/pepcore/pepcore_services/protein_expression/index.html (accessed April 16, 2012).